Multimodal optical contrast agents as new tools for monitoring and tuning nanoemulsion internalisation into cancer cells. From live cell imaging to in vivo imaging of tumours

Tailor-made NIR emitting dyes were designed as multimodal optical probes. These asymmetric amphiphilic compounds show combined intense absorption in the visible region, NIR fluorescence emission, high two-photon absorption in the NIR (with the maximum located around 1000 nm) as well as large Stokes' shift values and second-harmonic generation ability. Thanks to their structure, high loading into nanoemulsions (NEs) could be achieved leading to very high one- and two-photon brightness. These dyes were demonstrated to act as multimodal contrast agents able to generate different optical modalities of interest for bioimaging. Indeed, the uptake and carrier behaviour of the dye-loaded NEs into cancer cells could be monitored by simultaneous two-photon fluorescence and second-harmonic generation optical imaging. Multimodal imaging provided deep insight into the mechanism and kinetics of dye internalisation. Quite interestingly, the nature of the dyes was also found to influence both the kinetics of endocytosis and the internalisation pathways in glioblastoma cancer cells. By modulating the charge distribution within the dyes, the NEs can be tuned to escape lysosomes and enter the mitochondria. Moreover, surface functionalization with PEG macromolecules was realized to yield stealth NIRF-NEs which could be used for in vivo NIRF imaging of subcutaneous tumours in mice.


1
Synthesis of intermediate compounds 4-iodo-N,N-dibutylaniline (1) 1 : A solution of NaHCO 3 (2.68 g, 32.01 mmol) in 48 mL of water was added to a solution of N,N-dibutylaniline (4.00 g, 19.47 mmol) in 15 mL of CH 2 Cl 2 . The mixture was cooled down to 0°C and a solution of iodine (5.10 g, 20.37 mmol) in 225 mL de CH 2 Cl 2 was added dropwise, during 1 hour. The mixture was kept under stirring overnight at room temperature. The reaction was quenched with Na 2 S 2 O 3 (100 mL), and the organic phase was washed with Na2S2O3 (2*100 mL), water and dried over anhydrous Na 2 SO 4 . After filtration, the solvent was evaporated under vacuum and the residue was purified by silica-gel column chromatography using petroleum ether to afford 1 as a pale yellow oil (4.3 g, 67%). 1   and concentrated under vacuum. The oily residue was dissolved in 90 mL of toluene and the solution was refluxed under air bubbling for 16 h. After cooling down to room temperature, the mixture was filtered and the solid was collected and rinsed with toluene to afford 3 as a white solid (4 g, yield 57 %).

Monitoring of NEs uptake in U87 cells by confocal and two-photon imaging
The first image of the monitoring of the uptake of the NE[D1]-PEG by U87 cells was acquired 2 min after the addition of NE[D1]-PEG in cells imaging medium for 47 min with a time-lapse of 1.6 min between each image. For each snapshot, the bright-field, the two-photon imaging ( exc = 820 nm, 950 nm and 1020 nm) and the confocal imaging ( exc = 561 nm) were acquired (Movie S1).
The first image of the following of the uptake of the NE[D2]-PEG by U87 cells was acquired 7 min after the addition of NE[D2]-PEG in cells imaging medium for 47 min with a time-lapse of 1.5 min between each images. For each snapshot, the bright-field, the two-photon imaging ( exc = 820 nm, and 1020 nm) and the confocal imaging ( exc = 561 nm) were acquired (Movie S2).

Figure S6. SHG imaging on U87 cells incubated with NE[D2]-PEG for 1 h (A) and 2 h (B).
In order to confirm the origin of the fluorescence signal obtained by two-photon imaging we recorded the emission spectrum of the two-photon imaging of U87 cells at the end of the uptake experiment (i.e. 50 min of incubation time with NEs). The resulting emission spectra are shown in Figure S7 and clearly evidenced that the fluorescence signal is only due to the D1 and D2 dyes and not from auto-fluorescence background.

5.4
Ex-vivo fluorescence imaging Figure